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Publications (86)

  • Dong-Sun Lee · Clemens Vonrhein · Diana Albarado · [...] · Sudha Veeraraghavan
    [Show abstract] [Hide abstract] ABSTRACT: TEA domain transcription factors (TEAD) are essential for normal development of eukaryotes and are the downstream effectors of the Hippo tumor suppressor pathway. Whereas our earlier work established the three-dimensional structure of the highly conserved DNA binding domain using solution NMR spectroscopy, the structural-basis for regulating the DNA binding activity remains unknown. Here, we present the X-ray crystallographic structure and activity of a TEA domain mutant containing a truncated L1 loop, ΔL1 TEAD DBD. Unexpectedly, the three-dimensional structure of the ΔL1 TEAD DBD reveals a helix-swapped homodimer wherein helix 1 is swapped between monomers. Furthermore, each three-helix bundle in the domain-swapped dimer is a structural homolog of MYB-like domains. Our investigations of the DNA binding activity reveal that although the formation of the three-helix bundle by the ΔL1 TEAD DBD is sufficient for binding to an isolated M-CAT-like DNA element, multimeric forms are deficient for cooperative binding to tandemly duplicated elements, indicating that the L1 loop contributes to the DNA binding activity of TEAD. These results suggest that switching between monomeric and domain-swapped forms may regulate DNA selectivity of TEAD proteins.
    Article · May 2016 · Journal of Molecular Biology
  • [Show abstract] [Hide abstract] ABSTRACT: Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.
    Article · Oct 2015 · Nature
  • Article · Aug 2015 · Acta Crystallographica Section A: Foundations and Advances
  • Aaron D Finke · Ezequiel Panepucci · Clemens Vonrhein · [...] · Vincent Oliéric
    [Show abstract] [Hide abstract] ABSTRACT: Experimental phasing by single- or multi-wavelength anomalous dispersion (SAD or MAD) has become the most popular method of de novo macromolecular structure determination. Continuous advances at third-generation synchrotron sources have enabled the deployment of rapid data collection protocols that are capable of recording SAD or MAD data sets. However, procedural simplifications driven by the pursuit of high throughput have led to a loss of sophistication in data collection strategies, adversely affecting measurement accuracy from the viewpoint of anomalous phasing. In this chapter, we detail optimized strategies for collecting high-quality data for experimental phasing, with particular emphasis on minimizing errors from radiation damage as well as from the instrument. This chapter also emphasizes data processing for "on-the-fly" decision-making during data collection, a critical process when data quality depends directly on information gathered while at the synchrotron.
    Article · Aug 2015 · Methods in molecular biology (Clifton, N.J.)
  • Dong - Sun Lee · Diana Albarado · Clemens Vonrhein · [...] · Sudha Veeraraghavan
    [Show abstract] [Hide abstract] ABSTRACT: TEA domain (TEAD) transcription factors drive disparate developmental programs, including cell proliferation, cell differentiation, and organ size (Hippo pathway) in metazoans (TEAD1-4), as well as hyphal morphogenesis and biofilm formation in fungi (Tec1 / AbaA). Our earlier work (Anbanandam et al., PNAS 2006) revealed the tertiary structure of TEAD1 in solution and, through the use of protein binding microarrays, established the diversity in DNA binding sites recognized by this transcription factor. However, the molecular basis of DNA recognition by TEAD has remained elusive. Here, we will present the first X-ray crystallographic structures of TEAD-DNA complexes and of the deltaL1-TEAD variant defective in cooperative DNA loading. We will show the structural requirements for TEAD binding to single sites and cooperative recognition of tandemly duplicated sites. We will also discuss the striking similarities between TEAD and an unrelated transcription factor family, opening new vistas for probing signaling crosstalk. Finally, we will describe novel mechanisms for TEAD function gleaned from our single-molecule long-read mRNA sequencing data, and in hematopoietic stem cell expansion and energy metabolism.
    Conference Paper · May 2015
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    [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. Importance: Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is active only on RNA duplexes. We also show that pNS3h has a highly dynamic behavior--a characteristic probably shared with NS3 helicases from all Flaviviridae members--that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation or limit its dynamic range of conformations are indeed likely to block its helicase function.
    Full-text Article · Feb 2015 · Journal of Virology
  • Dong-Sun Lee · Diana Albarado · Clemens Vonrhein · [...] · Sudha Veeraraghavan
    Conference Paper · Jul 2014
  • Lee D-S · Albarado D · Vonrhein C · [...] · Veeraraghavan Sudha
    [Show abstract] [Hide abstract] ABSTRACT: The Hippo pathway is essential for the development of various organs in animals. TEA domain containing transcription factors are the downstream effectors of the Hippo pathway. Previously, we reported the solution NMR structure of the TEA domain (Anbanandam et al., 2006). Here, we discuss the first X-ray crystallographic structures of the TEA domain transcription factor (TEAD) complexed with double-stranded DNA. These structures identify requirements for the DNA-binding activity of the TEA domain. We then tested the structural insights using site-directed mutagenesis, gel shift assays, and solution NMR spectroscopy. Unexpectedly, our results have helped uncover a previously unrecognized mechanism in TEAD regulation. The findings reported here have implications for activity in muscles and hematopoietic stem cell development.
    Conference Paper · Feb 2014
  • Jun Hyuck Lee · Clemens Vonrhein · Gerard Bricogne · Tina Izard
    [Show abstract] [Hide abstract] ABSTRACT: The obligate intracellular, gram‐negative bacterium Rickettsia is the causative agent of spotted fevers and typhus in humans. Surface cell antigen (sca) proteins surround these bacteria. We recently reported the co‐localization of one of these proteins, sca4, with vinculin in cells at sites of focal adhesions and demonstrated that two vinculin binding sites directed the sca4/vinculin interaction. Here we report the 2.2 Å crystal structure of the conserved N‐terminal 38 kDa domain of sca4 from Rickettsia rickettsii. The structure reveals two subdomains. The first is an all‐helical domain that is folded in a fashion similar to the dimeric assembly chaperone for rubisco, namely RbcX. The following and highly conserved β‐strand domain lacks significant structural similarity with other known structures and to the best of our knowledge represents a new protein fold.
    Article · Aug 2013 · Protein Science
  • Jun Hyuck Lee · Clemens Vonrhein · Gerard Bricogne · Tina Izard
    [Show abstract] [Hide abstract] ABSTRACT: The obligate intracellular, gram-negative bacterium Rickettsia is the causative agent of spotted fevers and typhus in humans. Surface cell antigen (sca) proteins surround these bacteria. We recently reported the colocalization of one of these proteins, sca4, with vinculin in cells at sites of focal adhesions and demonstrated that two vinculin binding sites directed the sca4/vinculin interaction. Here we report the 2.2 Å crystal structure of the conserved N-terminal 38 kDa domain of sca4 from Rickettsia rickettsii. The structure reveals two subdomains. The first is an all-helical domain that is folded in a fashion similar to the dimeric assembly chaperone for rubisco, namely RbcX. The following and highly conserved β-strand domain lacks significant structural similarity with other known structures and to the best of our knowledge represents a new protein fold.
    Article · Jul 2013 · Protein Science
  • [Show abstract] [Hide abstract] ABSTRACT: The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways.
    Article · Jan 2013 · Cell
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    G. Bricogne · C. Vonrhein · P. Keller · [...] · P. Elliott
    Full-text Conference Paper · Aug 2012
  • Article · Aug 2012 · Acta Crystallographica Section A Foundations of Crystallography
  • C. Vonrhein · O. S. Smart · A. Sharff · [...] · G. Bricogne
    Article · Aug 2012 · Acta Crystallographica Section A Foundations of Crystallography
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    Ming Wei Chen · Masayo Kotaka · Clemens Vonrhein · [...] · Julien Lescar
    [Show abstract] [Hide abstract] ABSTRACT: The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.
    Full-text Article · Jun 2012 · Journal of bacteriology
  • Jun Hyuck Lee · Erumbi S Rangarajan · Clemens Vonrhein · [...] · Tina Izard
    [Show abstract] [Hide abstract] ABSTRACT: Vinculin is a key regulator of the actin cytoskeleton attachment to the cell membrane at cellular adhesion sites, which is crucial for processes such as cell motility and migration, development, survival, and wound healing. Vinculin loss results in embryonic lethality, cardiovascular diseases, and cancer. Its tail domain, Vt, is crucial for vinculin activation and focal adhesion turnover and binds to the actin cytoskeleton and acidic phospholipids upon which it unfurls. The RNA binding protein raver1 regulates the assembly of focal adhesions transcriptionally by binding to vinculin. The muscle-specific splice form, metavinculin, is characterized by a 68-residue insert in the tail domain (MVt) and correlates with hereditary idiopathic dilated cardiomyopathy. Here, we report that metavinculin can bind to raver1 in its inactive state. Our crystal structure explains this permissivity, where an extended coil unique to MVt is unfurled in the MVtΔ954:raver1 complex structure. Our binding assays show that raver1 forms a ternary complex with MVt and vinculin mRNA. These findings suggest that the metavinculin:raver1:RNA complex is constitutively recruited to adhesion complexes.
    Article · Jun 2012 · Journal of Molecular Biology
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    Thomas Krey · Francois Bontems · Clemens Vonrhein · [...] · Félix A Rey
    [Show abstract] [Hide abstract] ABSTRACT: Pestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site.
    Full-text Article · May 2012 · Structure
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    S D Yogesha · Erumbi S Rangarajan · Clemens Vonrhein · [...] · Tina Izard
    [Show abstract] [Hide abstract] ABSTRACT: The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of β-integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F-actin through the agency of its five-helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α-helices coined vinculin binding sites (VBSs) that reside within numerous four- and five-helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N-terminal seven-helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head-tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five-helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2-vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.
    Full-text Article · Apr 2012 · Protein Science
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    [Show abstract] [Hide abstract] ABSTRACT: Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct 'target' structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.
    Full-text Article · Apr 2012 · Acta Crystallographica Section D Biological Crystallography
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    [Show abstract] [Hide abstract] ABSTRACT: RuvBL1 (RuvB-like 1) and its homolog RuvBL2 are evolutionarily highly conserved AAA(+) ATPases essential for many cellular activities. They play an important role in chromatin remodeling, transcriptional regulation and DNA damage repair. RuvBL1 and RuvBL2 are overexpressed in different types of cancer and interact with major oncogenic factors, such as β-catenin and c-Myc regulating their function. We solved the first three-dimensional crystal structure of the human RuvBL complex with a truncated domain II and show that this complex is competent for helicase activity. The structure reveals a dodecamer consisting of two heterohexameric rings with alternating RuvBL1 and RuvBL2 monomers bound to ADP/ATP, that interact with each other via the retained part of domain II. The dodecameric quaternary structure of the R1ΔDII/R2ΔDII complex observed in the crystal structure was confirmed by small-angle X-ray scattering analysis. Interestingly, truncation of domain II led to a substantial increase in ATP consumption of RuvBL1, RuvBL2 and their complex. In addition, we present evidence that DNA unwinding of the human RuvBL proteins can be auto-inhibited by domain II, which is not present in the homologous bacterial helicase RuvB. Our data give new insights into the molecular arrangement of RuvBL1 and RuvBL2 and strongly suggest that in vivo activities of these highly interesting therapeutic drug targets are regulated by cofactors inducing conformational changes via domain II in order to modulate the enzyme complex into its active state.
    Full-text Article · Sep 2011 · Journal of Structural Biology